Universal fluorescent nanoprobes for one-step isothermal nucleic acid amplification assay

Abstract

The present invention provides a universal fluorescent probing system and method for result readout of loop-mediated isothermal amplification (LAMP) or reverse transcription-loop-mediated isothermal amplification (RT-LAMP) in a closed-tube manner using at least three different types of universal quantum dot probes, including sulfonate-, carboxyl-, and amine-modified quantum dots (QDs). Detection mechanism of the present system and method relies on a co-precipitation of the modified QDs with magnesium pyrophosphate crystals (one of the positive LAMP/RT-LAMP reaction products) formed during the reaction. The present system and method are advantageous over the existing technologies in terms of simple preparation, low cost, as well as excellent universality, specificity and sensitivity.

What is claimed is: 
     
         1 . A universal fluorescent probing system for one-step isothermal nucleic acid amplification assay, comprising a plurality of functionalized fluorescent nanoprobes capable of forming co-precipitates in the presence of positive isothermal amplification reaction products along with a target sequence amplification. 
     
     
         2 . The system of  claim 1 , wherein the plurality of functionalized fluorescent nanoprobes comprises one or more semiconductor quantum dots (QDs) with one or more surface modifications. 
     
     
         3 . The system of  claim 2 , wherein the one or more semiconductor QDs comprise cadmium selenide sulfide/zinc sulfide (CdSeS/ZnS core/shell) quantum dots. 
     
     
         4 . The system of  claim 2 , wherein the one or more semiconductor QDs are surface modified by one of 2-mercaptoethanesulfonate, mercaptoacetic acid, and cysteamine to result in sulfonate-QDs, carboxyl-QDs, and amine-QDs, respectively. 
     
     
         5 . The system of  claim 4 , wherein the positive isothermal amplification reaction products with which the plurality of functionalized fluorescent nanoprobes is capable of forming coprecipitates include magnesium pyrophosphate (Mg 2 P 2 O 7 ) crystals. 
     
     
         6 . The system of  claim 5 , wherein the sulfonate-QDs or carboxyl-QDs form coprecipitates via complexation between Mg 2+  and P 2 O 7   4−  of the Mg 2 P 2 O 7  crystals with the sulfonate or carboxyl group of the sulfonate-QDs and carboxyl-QDs in the course of the isothermal amplification reaction. 
     
     
         7 . The system of  claim 4 , wherein the amine-QDs form coprecipitates via electrostatic attraction between P 2 O 7   4−  of the Mg 2 P 2 O 7  crystals and amine group of the amine-QDs in the course of the isothermal amplification reaction. 
     
     
         8 . The system of  claim 1 , wherein the isothermal amplification reaction comprises loop-mediated isothermal amplification (LAMP) and reverse transcription-loop-mediated isothermal amplification (RT-LAMP) reactions. 
     
     
         9 . The system of  claim 8 , wherein the LAMP reaction is performed at about 65° C. for 1 hour or less. 
     
     
         10 . A method for closed-tube detection of a target nucleic acid sequence in a sample based on an isothermal amplification reaction, the method comprising:
 providing the plurality of functionalized fluorescent nanoprobes of the universal fluorescence probing system of  claim 1  to an isothermal amplification reaction mixture containing the sample;   incubating the plurality of the functionalized fluorescent nanoprobes with the isothermal amplification reaction mixture containing the sample at an isothermal amplification temperature for a time interval;   analyzing the isothermal amplification reaction mixture during or after said incubating by a fluorescence reader, wherein supernatant portion of the reaction mixture is analyzed; and   determining the presence of the target sequence in the sample by detecting any fluorescence precipitates formed in the isothermal amplification reaction mixture during or after said incubating;   otherwise, detecting a fluorescence dispersion in the isothermal amplification reaction mixture after said incubating for verifying the absence of the target sequence in the sample.   
     
     
         11 . The method of  claim 10 , wherein the isothermal amplification reaction mixture comprises isothermal amplification reagents capable of forming Mg 2 P 2 O 7  crystals when positive amplicons of the target sequence are generated by either LAMP or RT-LAMP reaction. 
     
     
         12 . The method of  claim 10 , wherein an analyte of the sample comprises one or more types of nucleic acids directly obtained or isolated from a test subject, object, or biological sample. 
     
     
         13 . The method of  claim 10 , wherein the isothermal amplification temperature is about 65° C., and the time interval for said isothermal amplification is about 1 hour or less. 
     
     
         14 . The method of  claim 11 , wherein the LAMP or RT-LAMP reaction is carried out in any device with uniform heating and cooling capabilities and a time control mechanism. 
     
     
         15 . A method of fabricating the universal fluorescence probing system of  claim 1  for an isothermal amplification reaction, the method comprising:
 selecting a semiconductor quantum dot; 
 modifying surface of said semiconductor quantum dot with one or more functional groups such that the one or more functional groups are capable of interacting with Mg 2+  and/or P 2 O 7   4−  to form coprecipitates. 
 
     
     
         16 . A kit for detecting a target nucleic acid sequence in a sample, the kit comprising any one or more of the functionalized fluorescent nanoprobes of the universal fluorescence probing system according to  claim 1 , reaction components for LAMP or RT-LAMP reaction including isothermal amplification buffer, deoxynucleotides, enzymes, betaine, LAMP primers with respect to the target sequence, stabilizers or additives for facilitating formation of the coprecipitates, a control sample, and solvent or solution for dissolving any of the reaction components.